The post genomic era promises to revolutionize science and medicine through a detailed understanding of biology at the molecular and system levels. As our fundamental knowledge of the underlying biochemical causes of various human ailments rapidly improves, an ever increasing emphasis will be placed on personalized treatments, better monitoring of patients during the course of therapy, and point-of-care diagnostics. Advances in these emerging fields will depend on biosensor technologies that are portable, easy to use, and can display high detection sensitivity, accuracy, analysis speed, and throughput. Here we propose a basic research program in biomolecular sensing that is inspired by the functioning physicochemical principles of intrasterically regulated natural enzymes. The proposed biosensor element is modular in design and is based on specific covalent attachment of an enzyme to its small molecule inhibitor through an allosteric tether. Upon interaction of the tether with its complement (nucleic acid, peptide, or protein), the ensuing conformational change activates the enzyme by liberating the inhibitor from its active site. The activated enzyme complex, through substrate turnover, serves as a built-in signal amplifier of the molecular recognition event. The utility of this novel concept has already been established in the design of a highly sensitive one step detection of specific DNA sequences (Preliminary Results section). The proposed research program seeks to further advance and optimize this new sensing methodology. The specific aims of the proposed research program are: I. To design and characterize improved variants of the Inhibitor-DNA-Enzyme (IDE) chimera through rational modifications of the enzyme attachment site, DNA tether structure and size, and the covalent structure of the inhibitor. II. To design IDEs for rapid detection of biologically relevant genetic markers and single nucleotide polymorphism (SNP). III. To explore the generality of the intrasterically regulated enzyme chimera in protein detection by engineering an Inhibitor-Peptide-Enzyme (IPE) chimera for recognition of GP-41 in the HIV virus.